Wednesday, 11 December 2013

Primary Cells - the Definition!



Hi all,

Today I've got a key definition for all those of you who are culturing primary cells, or may be thinking about it!

What is a primary culture?  Here's a formal definition:

A primary culture is the stage after the isolation of the cells from tissue, but before the first sub-culture.  (At the point of the first sub-culture, it becomes a cell line).

In practice it is often very important to clarify this definition, since the widespread commercial availability of human primary cells has led to frequent alternative use of the terms "primary cells" or "primary cell lines".  This can be very misleading.

In some cases, you may obtain cells from a supplier that are labelled "primary cells".  These are (or should be!) cells that have been cryopreserved at the end of the primary culture stage.  When you thaw these into culture, they become a finite cell line, beginning at passage number 1.  Each time they are sub-cultured, the passage number increases by 1 (passage 1, passage 2, passage 3 etc).

However, in other cases, you may receive cells that are referred to as "primary cells" but they have actually gone through a number of sub-cultures prior to arrival in your lab - in this case they are not actually primary cells, but are already a finite cell line.  Be sure to check the passage number and ask the supplier if you need clarification.

Clarification of the culture stage is important because it affects the uniformity of the cell population and the number of generations remaining before senescence (ie the lifespan).  This could have a serious impact on the performance of your cells during the course of your experiments.  Starting experiments with older cells could even mean that the culture starts to senesce and die before you've completed your work (remember these are finite cell lines with a short lifespan).

Note that, technically speaking, if cells are subcultured from a true primary culture, the correct term is "early passage cell line" and not a "primary cell line", which is a contradiction in terms!!

Remember: all cell cultures begin as primary cells, even if they are subsequently treated to become finite or immortal cell lines.  So primary cells are important as the source of all cell cultures!

More later this week about primary cells vs. cell lines, and how to make the most of the short lifespan of your finite cells!  Meanwhile, please feel free to contact us by leaving any questions or comments here, email info@x-cellr8.com or via Facebook at https://www.facebook.com/XCellR8

Primary fibroblasts in culture, isolated from the dermis of human skin. 
These cells are used at XCellR8 to perform in vitro efficacy testing to support cosmetic claims
such as collagen-boosting, anti-oxidant and anti-ageing:

Happy Cell Culturing! :)
Cellwyn
 

Friday, 29 November 2013

Happy Thanksgiving to all our American friends!


Hi all!

Just a special note to wish all of our American friends a very Happy Thanksgiving holiday weekend!  Quite a few of our blog followers are based in the US, so we'd like to say a big thank you for your support!  It's business as usual here in the UK, but we always have a little time out for a break and here I am catching up with Dr Carol Barker-Treasure, XCellR8's founder and a good friend of mine!  Have fun :)
Cellwyn and all at XCellR8
PS We are hoping to bring some of our cell culture training events to the US in the near future.  If you think you or your colleagues may be interested, drop us a line at info@x-cellr8.com.  Check out our training events at http://www.x-cellr8.com/training.aspx

How often should you re-feed your cells?


Hi everyone!

Do you have plans for the weekend, and are wishing that you didn't have to go to the lab to re-feed your cells?

Here's a general rule that can be applied for many adherent cultures:
If your cells are less than 50% confluent (covering less than 50% of the culture surface area), they will usually be happy with a re-feed every 72 hours (that means they'll last from Friday to Monday - woohoo!)  However, if they are more than 50% confluent, they will usually require a re-feed every 48 hours to keep them in optimal health.  As there are more cells present, they are collectively using up more nutrients.  (That means you should pop in over the weekend to do a re-feed).

Consider sub-culturing your cell lines on a Friday so that they are at low confluency over the weekends and therefore won't require re-feeding until Monday. 

We know you all love your cells, and giving them all the TLC they need is essential, but it's important to have your time off too!  So it's worth being smart about your sub-culturing schedule!

Please do keep in mind that every cell type is different, and if you work to a quality compliance system such as GLP or ISO in your lab, don't forget to stick to your SOPs or ask your manager if you'd like to suggest a change!

Do you have a question about this post or about Good Cell Culture Practice in general?  Email us at info@x-cellr8.com or visit our website to find out about our upcoming training courses:
http://www.x-cellr8.com/training.aspx

Have a fantastic weekend - whether you are re-feeding your cells or not!

Cellwyn

Wednesday, 20 November 2013

Cell Counting - some useful information

Hi all,

Here's some really useful information that we received today about cell counting, that could make life easier if you are working with samples containing debris.

We're interested in your feedback and experiences!  Leave your comments here or on our Facebook page: https://www.facebook.com/XCellR8

Have a great day :)
Cellwyn

Trypan Blue vs AOPI – How to Choose the Optimal Viability AssayUsing Trypan Blue and Fluorescent AOPI Dyes to Measure Cell Viability by Cellometer Cell Counters
question
Do you have red-blood cells, cell debris, or tissue debris in your samples?
Here are some samples that may contain RBCs, cell and tissue debris, and have been analyzed by the Cellometer instruments:
 
 
·        Tail Vein Blood
·        Splenocytes
·        Bronchoalveolar lavage
·        Tumor Digests
·        PBMC
·        Lymphocytes
·        Cord Blood
·        Peripheral Blood
·        And many more
 
check
When your samples contain unwanted particles use:
Acridine Orange/ Propidium Iodide (AO/PI) viability assay to accurately measure the concentration and viability of your samples!
 
fbfc
 
vdv
AO/PI Protocol
1.     Obtain Nexcelom AO/PI solution: Cat # CS2-0106-5ML
2.     Stain cell sample at 1:1 with AO/PI solution
3.     Load counting chamber slide with 20 µl of cells and analyze

Accurate and Consistent Results in <30 seconds!
 
To determine which instrument is best for you, contact a Nexcelom specialist today!
Ask a Cellometer Applications Specialist

question
Do you have clean cell samples without RBCs, or other cell debris?
Here are some samples that may be considered clean samples, and have been analyzed by the Cellometer instruments:
 
 
·        Over 1600 cultured cell lines
·        All NCI 60 cancer cell lines
·        Isolated Stem Cells
·        Isolated Mononuclear cells with RBC lysing
·        Frozen clinical Samples
·        Isolated Primary Cells
 
check
When your samples are free from unwanted particles use:
Trypan Blue exclusion assay to accurately measure the concentration and viability of your samples!
 
fbfc
 
vdv
Trypan Blue Protocol
1.     Dilute the stock (0.4 %) with PBS to 0.2 %
2.     Filter the trypan blue with 0.2 micron filter
3.     Mix the cell suspension at 1:1 with 0.2 % trypan blue
4.     Load the counting chamber slide with 20 µl of cells into the Cellometer and analyze

Accurate and Consistent Results in <30 seconds!
 
To determine which instrument is best for you, contact a Nexcelom specialist today!
Ask a Cellometer Applications Specialist

Monday, 16 September 2013

Win £300 in FRAME's essay competition

Hi everyone!
Are you aged 16-19 and living in the UK?
You could win £300 by entering FRAME's essay competition!
The theme of the competition is "What are the alternatives to using animals in laboratories"?
You've got some time to get your thinking caps on as the closing date isn't til 12 January 2014.  For a few ideas for starters, why not check us out on Facebook, where we post news stories about new alternatives to animal testing?
https://www.facebook.com/XCellR8?ref=hl
I might have a go myself :)
http://www.frame.org.uk/news_details.php?news_id=176
Catch up soon - myself and Lottie will be back with more news and cell culture tips!
Cellwyn

Friday, 6 September 2013

Training in the Lab - Observing the Cells

Bushra has been training me on the importance of observing the cells every day, to ensure they are happy and healthy, and to check that there is no contamination! The cells we thawed and cultured are looking healthy and are growing well. 
The Balb:c 3T3 Cells after 24hours in the incubator
I am glad to see the cells are growing well and am looking forward to watching them grow more over the next few days. 
Lottie

Thursday, 5 September 2013

Training in the Lab - Thawing the Cryopreserved Cells and Establishing a Culture

Over the past few days at XCellR8, I have been training and learning how the Balb/c3T3 Cells are cultured. 

Bushra started by showing my step-by-step how each stage of the cell-culture process is carried out. 
Firstly the cryopreserved cells are removed from the liquid nitrogen and then rapidly thawed in the water bath.


During the demonstration I learnt about important aseptic techniques, such as ensuring the ampoule is not fully immersed in the water bath, as this could increase the risk of contamination.


After the cells have been thawed, the contents of the ampoule is centrifuged to separate the cells from the supernatant. I learnt about how to counterbalance the centrifuge and the importance of adding culture medium to the cell suspension to ensure the cells survive!


After the cells have been centrifuged, they are resuspended in medium, and a cell count is performed. A cell count is performed using a haemocytometer and trypan blue exclusion stain. This part looked quite confusing and I think it will take some practice before I perfect it!



After the concentration of cells has been determined, the cells are seeded at the recommended density in a T25 flask. The flask is then placed in the incubator which provides the perfect conditions for the cells to grow.


I am looking forward to revisiting the cells over the next few days and seeing how they grow!
Lottie 



Thursday, 29 August 2013

First Impressions

Today, Bushra showed me around the lab where all the XCellR8 work takes place. She talked me through all the different equipment in the lab, and what it was for. I was particularly interested in the Class II Safety Cabinet, which provides a sterile environment in which to work. I look forward to learning how to use this over the next few days, and to bettering my aseptic technique.

Bushra, demonstrating the safety cabinet
Looking forward to another day tomorrow!
Lottie 

Tuesday, 27 August 2013

Introductions

Hi, I'm Lottie and I am a Biology student at MMU. I have recently started a summer placement at XCellR8, and over the next few days, I will be guest-blogging about my experiences at XCellR8 and what I have learnt. 


Wednesday, 21 August 2013

Have you checked us out on Facebook?

Hi everyone!
Our Facebook page now contains loads of interesting updates and news about cell culture, skin, cosmetics testing and the latest developments in alternatives to animal testing.  Connect with us there, join in the fun and learn something new!
https://www.facebook.com/XCellR8
Coming soon on Facebook: competitions to win a free place at our training events!
And coming soon on this blog: my very own hints and tips on Good Cell Culture Practice.
Have a great day :)
Cellwyn

Thursday, 25 July 2013

Special message for students!

Hi all!

Hope you're enjoying this gorgeous summer weather as much as I am! 

 
Today I've got a special message for students!  Here at XCellR8, we are developing a programme of cell culture training activities specifically designed for students, as a valuable addition to all the educational stuff you're already learning at University - either as an undergraduate or postgraduate in the Life Sciences.  We will be highlighting the scientific benefits as well as the ethical advantages of using human cell culture as a valuable alternative to traditional animal test methods.  We'll be discussing lots of technical aspects and practical advice in terms of Good Cell Culture Practice, as well as direct applications of the technology in industries such as pharmaceuticals, biotechnology, cosmetics, personal care and healthcare.  All participants in these workshops receive a Certificate of Attendance and a few classic mementos including a Cellwyn badge :)  Students who have attended these events in the past have given great feedback and have benefited in terms of increased knowledge and an enhanced CV.  If you're interested in finding out more, please register your interest by emailing info@x-cellr8.com.  It's likely that the next event will take place in the Manchester area in early November, once you've all settled back into the new term.  However we're thinking of doing additional events at other locations, so please do register your interest and we'll base our plans on your feedback.
 
Look forward to hearing from you!
Cellwyn

Monday, 10 June 2013

Upcoming Conferences


Hi everyone,

Just thought I’d let you know that a couple of conferences have recently been announced that might be of interest to you:

The 9th World Congress on Alternatives and Animal Use in the Life Sciences: Prague, 24-28 August 2014:
http://www.wc9prague.org/
This is the perfect place to learn more about the current status and future challenges of developing and implementing in vitro alternatives to traditional animal tests.  The topic in Prague will be “Humane Science in the 21st Century” and will cover a broad range of subjects. 

Also, the European Society for Toxicology In Vitro (ESTIV) has recently announced its next international conference, taking place in Egmond aan Zee, The Netherlands, 10-13 June 2013.  The topic will be “Making Sense of In Vitro Methods” and scientists from across Europe and beyond will be doing just that!

I’m already packing my bags and looking forward to a bit of European travel next year, to take part in these great events J

Thursday, 6 June 2013

Cell culture as a true replacement for animal testing......................

Hello friends!
 
This week, I’ve been learning about the use of cell cultures to test chemical toxicity.  This is an important topic right now, as industry prepares to meet the deadlines associated with the REACH legislation.  In particular, the widely used Balb/c 3T3 cell line brings up some interesting points about the extent to which cell culture systems can be considered truly “in vitro”.


 
Balb/c 3T3 clone A31 fibroblasts, 72 hours post seeding

As a bit of background, the cells were originally isolated from a single mouse embryo back in 1968.  They were continuously cultured according to a strict regimen and underwent spontaneous transformation, meaning that they can now be cultured indefinitely.    [For the purpose of Good Cell Culture Practice, XCellR8 adopts a maximum passage number (p100) at which the cells may be used in our laboratory, to ensure optimal data quality – and we would recommend this practice].  Balb/c 3T3 fibroblasts are cultured in DMEM supplemented with foetal bovine serum (FBS) and L-Glutamine.  So they are animal cells, cultured using some animal-derived products.

There's no doubt that cell culture technology has enabled huge advances in terms of replacing animal tests over the past few decades.  However, sometimes it's still necessary to use animal derived cells, media components and reagents, such as Balb/c 3T3s cultured in serum-containing medium.  Sometimes, industry is forced into doing this because the current OECD Test Guideline for a particular test may cite the use of an animal cell line.  To produce safety data in compliance with regulations, the OECD published protocol must be adhered to.  One example is OECD Test Guideline 129 (Acute Toxicity), which utilises the Balb/c 3T3 cell line and the Neutral Red Uptake (NRU) assay to predict the cytotoxic potential of chemicals. 

Scientifically, there is an alternative: it is possible to carry out the same cytotoxicity test using human cells (eg epidermal keratinocytes or dermal fibroblasts) cultured in serum-free media.  Even though this involves some additional cost, it improves the ethical value of the test, making it truly “in vitro”.  Here at XCellR8 we would like to see a new protocol for the Acute Toxicity test, using human fibroblasts in serum-free medium, validated for acceptance as a new OECD test guideline.

We also have a policy of using human cells and completely animal product-free culture systems wherever it is possible to do so.  In recent years there have been lots of advances in developing serum-free and chemically defined culture media and reagents.  We’d like to encourage the wider use of these systems and there’ll be more info to follow. 

What do you think about these issues?  Feel free to add your comments here or email us at info@x-cellr8.com.

Catch up again very soon!
Cellwyn



Thursday, 30 May 2013

New lab!

Hi all,

You may have been wondering why I haven't posted anything for a while - but I am still here to entertain you! :)

It's been a really busy few months here at XCellR8, not least because we've moved to fantastic new premises at Sci-Tech Daresbury in Cheshire.  The move will allow us to accommodate our customers' requirements for our in vitro testing services and cell culture training events.  We're in a great location between Manchester and Liverpool, easily accessed by plane, train and automobile!  If you're interested to read more, click here

Here's a photo of me settling into the new premises, looking rather handsome ;)


Now that we're fully installed I'll be posting regular updates here including cell culture hints and tips, news about new developments in the industry and advances in alternatives to animal testing.  So please do check back, feel free to follow my blog and join in by posting your comments!

Cellwyn

Thursday, 10 January 2013

Happy New Year!


 
Hi everyone!  First of all, a very Happy New Year!  I hope that 2013 turns out to be a fantastic year for you! 
It’s certainly going to be a great year, and a busy one, here at XCellR8.  We are making preparations for a range of new and exciting cell culture training activities, as well as our regularly scheduled events.  Do you have a wish list of training requirements?  If so we’d love to hear from you.  We even offer customised events on-site at your premises, including Good Cell Culture Practice seminars and updates for your team, so feel free to contact us for a free informal consultation.  I may even make a guest appearance with my colleagues J