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Author: Subject: Extraction of human stem cells from blood (And another question)
Nate
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[*] posted on 3-6-2018 at 17:22
Extraction of human stem cells from blood (And another question)


1. Hello, I have recently become enamored with human t-cells and (blood)stem cells. The only way I know how to extract these cells is with the RosetteSep system(Links to which are below), but this system is quite obviously expensive. I was looking for a cheaper alternative if anyone knew one.

2. I've also become quite delighted by phages. I love the science behind them and find their medical promise interesting. I would like to see if I could genetically modify them using the CRISPR system, what I would edit these phages to do I am not sure, but the idea interests me. I know using the CRISPR system to edit phages is possible because I've read a couple papers(links below) on it but I'd like to hear how others would do this.

Links:
(papers):
http://mmbr.asm.org/content/80/3/523.full
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5553622/
(RosetteSep)
https://www.stemcell.com/products/product-types/cell-isolati...

Thank you in advanced!

(Sorry for the bad writing, I know I could've worded this better but I wrote this quickly)
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[*] posted on 3-6-2018 at 17:52


There are several different approaches for isolation of specific cell populations from whole blood. Including several on the page you linked to. RosetteSep is not one I had heard about but does seem quite elegant.

Many procedures will begin by separating peripheral blood mononuclear cells (PBMCs) from the red blood cells and plasma. The specific population is then purified. For some cell types there are relatively low cost ways of purifying but I don't know of any T cell protocols that don't rely on antibodies. Antibodies always increase the cost and this is a real impediment to any sort of self-study / amateur experimentation.

Antibodies bind to a protein on the surface of the cells. The two main approaches are paramagnetic beads and cell sorting. The antibodies are labeled / attached to paramagnetic beads and pulled down by a strong magnet, bringing the cells with them. They can also be attached to a fluorophore and the cells separated by fluorescence activated cell sorting (FACS).

What would you want to do with them when you have them?
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Nate
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[*] posted on 3-6-2018 at 18:36


I do not have an immediate use for stem or t-cells, I just thought it'd be cool to see if I could do it. Although, I have found out that stem cells can be used to replicate other bodily cells which I thought would be cool to try(if I can of course). May you also elaborate on how these cultures are purified? I'm relatively new to biology in general. :P

[Edited on 4-6-2018 by Nate]
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streety
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[*] posted on 4-6-2018 at 15:35


Stem cells are present at only low levels in the blood. They are there but if you wanted a lot of cells for a bone marrow transplant for example it's not a practical solution. More details on the two main approaches for that can be found at https://www.anthonynolan.org/8-ways-you-could-save-life/dona...

Isolation of the cells would be very similar to T cells. The main difference would be using antibodies that bind to different receptors, for example CD34 for the stem cells instead of CD4 or CD8 for T cells. Although those receptors (CD4 and CD8) might be avoided to prevent activating the cells.

If you are interested in how different tissues could be replicated/grown you might be interested in induced pluripotent stem cells. These can be created from any adult cell and can be directed to differentiate into many different types of tissue.
https://en.wikipedia.org/wiki/Induced_pluripotent_stem_cell
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Nate
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[*] posted on 4-6-2018 at 20:31


Well, in theory even if I did get a tiny amount of stem cells they could reproduce themselves correct? (Provided the right criteria of course)

[Edited on 5-6-2018 by Nate]
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streety
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[*] posted on 5-6-2018 at 03:18


Yes, that's correct. Their growth and maintenance is quite involved though.
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Nate
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[*] posted on 5-6-2018 at 07:08


As I assumed, can you specify some of the methods on how to grow stem cells in a lab artificially?
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[*] posted on 5-6-2018 at 08:21


I've done a few CRISPRs on fruit flies; it's pretty straightforward and I've included the protocol that we basically followed. Based on your interests you might want to try to get access to Current Protocols in Molecular Biology through a local library/university/SciHub/the References subforum.


Attachment: Crispr CAS9 editing in Drosophila (874kB)
This file has been downloaded 118 times

You'll need a moderately functional molecular biology lab (there are a few DIY Bio resources on and off this site; you might look up user chironex and their YT channel: https://www.youtube.com/channel/UCV5vCi3jPJdURZwAOO_FNfQ ). You'll also need some basic bioinformatics tools. Designing the guide RNAs requires finding appropriate PAM sites in the genome as well as checking for "off target" effects in which the edited sequence is not unique enough to prevent other parts of the genome from also being edited. Sometimes sites without off-target effects can't be found, and you have to settle for an intronic or intergenic off-target, hoping that it's silent.

I used these fly-specific resources, but it's probably not hard to find or write similar software for your organism of interest:

http://tools.flycrispr.molbio.wisc.edu/targetFinder/

http://flybase.org/cgi-bin/gbrowse2/dmel/

One thing about viruses is that they usually (though not always ) have relatively small, compact genomes, which might have consequences for finding editing sites: there will be fewer PAM sites to choose from, which might mean less freedom to edit, but also fewer off-target effects, but the off-targets might be more likely to be non-silent in a gene-dense genome... I dunno, it would probably depend on the specifics of what you're doing.







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Nate
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[*] posted on 5-6-2018 at 09:34


Thank you for the insight mayko! While I'm here I'd also like to ask if it's possible to make the antibodies needed to isolate stem cells outside of the body or isolate it from the body itself. Sorry if this is a stupid question like I said I'm new to biology in general so I'm going to ask as many questions as I can.
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[*] posted on 5-6-2018 at 16:52


Antibodies are produced by the immune system so unfortunately require an animal. Mice, rats, rabbits, goats, sheep, etc are all popular options. After an antibody has been found though its production can be moved to an in vitro system (i.e. cell culture without any animals used).

There are other options though that could potentially be used to produce antibody alternatives. Aptamers and Affimers are two options.
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[*] posted on 5-6-2018 at 22:01


How and where would I find the antibodies? The lymph nodes?
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[*] posted on 5-6-2018 at 23:57


They are usually isolated from the blood.

There are several ways to isolate the antibodies, with varying levels of difficulty and resulting in different levels of purity.

You'll probably need very pure antibodies for your purpose, which means that you'll probably have to use a highly selective method such as affinity chromatography for your last step. Basically, you bind your antigen to a solid substrate, fill a chromatography column with that, pass the antibody-containing preparation through it to bind your antibody, wash all the other stuff away, and finally elute your pure antibody.

Before you get to that step, you will have to perform some initial purification steps to get rid of the bulk of the other stuff in the raw serum though, such as ammonium sulphate precipitation and perhaps an isolation of the IgG with protein A.

As an alternative to this route for isolating stem cells, I suggest you study the methods that were first used in the early days of stem cell research. A particularly easy and successful method exploits the fact that the mitochondria of heamatopoetic stem cells do not take up rhodamine-123.
You can label the cells with this stuff, and then select the cells that do not fluoresce. You would need a cell sorter for it though.

I must say you have set yourself quite an ambitious goal for your first project.




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[*] posted on 6-6-2018 at 07:13


Yeah, it is quite an "interesting" one to say the least. I do have another question, as always. Regarding the rhodamine-123 method you provided above, instead of using a cell sorter could I isolate a few cells manually and then force them to grow in a culture flask?

Edit: By using a blacklight or other UV source to make them fluoresce.

[Edited on 6-6-2018 by Nate]
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[*] posted on 6-6-2018 at 08:24


In theory, yes.
However, you will need a fluorescent microscope and maintaining sterility could be a practical issue.
Remember you'll also need a humidified, air / CO2 incubator with appropriate air tanks and a laminar flow hood for this type of work.
Other consumables include tissue culture media, antibiotics/antifungal solutions, and sterile pipettes, culture flasks , etc.
I haven't performed any tissue culture work in over 30 years but stem cell isolation and culture is not a trivial task. Suggest you try to visit a lab where someone is actually doing this work to see what you need and what's involved.
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[*] posted on 6-6-2018 at 08:48


Good idea! I'll look for some labs in my area. I'm aware that I need a CO2 incubator and am currently trying to come up with ways of either buying one or making one. As for the laminar flow hood, is it possible to make one as well? I'm sure it is but I'd need to do some research.

Edit: Trying to make as much of the things I need so that I can to limit the price, though I'm expecting this to be an expensive project.

Edit: So is making a DIY fluorescence microscope doable as well? I find a pdf file about it, link below. The article mentions the use of a 3d printer which surprisingly my friend owns one :D. Also, a follow up to the Laminar flow hood question I asked I found an article on that too.

http://www.instructables.com/id/Portable-Laminar-Flow-Hood/
https://pubs.acs.org/doi/pdf/10.1021/acs.jchemed.5b00984 (Didn't read all of it, but most of it)

[Edited on 6-6-2018 by Nate]

[Edited on 6-6-2018 by Nate]

[Edited on 6-6-2018 by Nate]

[Edited on 6-6-2018 by Nate]
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[*] posted on 6-6-2018 at 11:15


Yes, you could in principle sort them manually with a fluorescence microscope, but it will be very time consuming. In bone marrow, only 1 in 2,000 to 1 in 10,000 cells is a haemopoetic stem cell. In blood, they are much rarer even.

A laminar flow hood is not very complex. It should certainly be possible to build one. Basically, you need to pump air through a HEPA or ULPA filter to sterilise it and then pump it into the flow cabinet at a high enough rate (but without turbulence, and distributed through the cabinet) that air only exits but does not enter the flow cabinet. In that way, you can maintain a reasonably sterile environment to work in.

MountainMan's suggestion is a very good one. Culturing a simple cell line, one that is a little bit forgiving, can easily be taught in a few hours. If you already have some lab experience, an hour is enough already. It is not very difficult, but much easier learned from watching someone and then doing it yourself than from a text-based forum. Once you got that down, you can expand your skills working with cells that are more difficult to maintain.


[Edited on 6-6-2018 by phlogiston]

[Edited on 6-6-2018 by phlogiston]




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[*] posted on 6-6-2018 at 13:46


Can you give an example of a "simple cell line"? Like I said I'm still unfamiliar with some of these terms. Also, I did find a lab nearby that looks promising for this kind of work.
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[*] posted on 6-6-2018 at 16:51


"Simple cell line" are my words, not any officially recognised term :)
I would consider a 'simple cell line' one that:
- will grow in most common media and does not have any unusual requirements, for example with respect to serum added.
- does not differentiate
- grows fairly quickly
- is immortal/continous.

As an example, HEK293 cells are pretty easy to work with.

Gibco (a well-known supplier of cell culture supplies) has a nice guide that describes many basic techniques and theory.
Googling, I found a copy here amongst other places, but I don't know if it is the original online source of the document:
https://www.thermofisher.com/content/dam/LifeTech/latin-amer...




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[*] posted on 6-6-2018 at 19:42


Thank you so much! I will continue to look into the reproduction of stem cells in an amateur lab but this is a great way for me to get started in this field in the meantime. I'll still post my questions here though since like I said, I'm still new to this field in general.

Edit: Happen to know a cheaper place to buy (HEK293 cells) them? Or if they sell them for cheaper, I don't have a lot of money. Here's a link the ones I found:
https://www.thermofisher.com/order/catalog/product/11631017?...


[Edited on 7-6-2018 by Nate]
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[*] posted on 7-6-2018 at 08:50


I've been researching a little on my own and now and I have a couple more questions, is it easy to grow a "skin cell" culture? I've also found out that you can revert skin cells back into stem cells, how is this done and is it possible to do in a home laboratory? If I could grow this skin cell culture, what could I do with it afterward, other then turning them into stem cells of course.

Edit: After reading one of the links you guys provided for me on iPCS, I got the understanding that stem cells could be made from a single pluck of hair, or a certain cell type from urine("obtaining fibroblasts to produce iPSCs involves a skin biopsy, and there has been a push towards identifying cell types that are more easily accessible. In 2008, iPSCs were derived from human keratinocytes, which could be obtained from a single hair pluck. In 2010, iPSCs were derived from peripheral blood cells, and in 2012, iPSCs were made from renal epithelial cells in the urine.")
https://en.wikipedia.org/wiki/Induced_pluripotent_stem_cell

[Edited on 7-6-2018 by Nate]
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Nate
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[*] posted on 7-6-2018 at 21:38


Okay, I believe I have found a way to make iPSCs but need some confirmation of some things. 1. The 4 proteins/factors needed to turn a bodily cell into a PSC are Oct4, Sox2(this is a gene I believe), Kfl4, and c-Myc. Furthermore, I have found evidence that only one of these genes or proteins/factors needs to turn the cell back into an embryonic state(Supported by:https://www.cell.com/cell-stem-cell/fulltext/S1934-5909(17)30501-5). The information I want/need is the gene I need to "knockout" to active the Sox2 gene, I will be using Synthego so gain access to CRISPR technology (Because it gives you a free trial :D). Here is a link to Synthego if you want to check it out yourself; https://tools.synthego.com/#/. Thanks in advanced :D.
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[*] posted on 30-8-2018 at 04:09




Nate, I have researched extensively (a long time ago) into how to acquire stem cells. I have no idea how someone can acquire the genes like Oct4, Sox2, Kfl4, etc.... but a reasonable alternative is to take a look at MM-401. It is a peptide type of chemical that singlehandedly converts cells to stem cells. I believe they may also be"naive" pluripotent, which is better than just normal pluripotent... or something. It's been a while... Anyways, I never had the chance to look into how to make this peptide and it was too expensive to. You could ask for a price quote from:

https://medkoo.com/products/17840

...and let us all know the price per gram, if you're interested. I also took a look at "apheresis" I believe it's called. This type of machine simply centrifuges the blood, and removes the stem cells, then puts the blood back into the patient. I think the problems with this were, some kind of apparatus for removing the stem cells being expensive, but I can't remember, they may just need to be siphoned out. Another problem MIGHT have been that these kinds of stem cells might not be able to turn into any cell other than various types of blood cells. It's been years since I've read into this stuff, sorry.

Yea the problem with HEK cells is that they have to be stored at cryogenic temperatures. Universities have a limit on how many of these cryo things they can have because they cost insane amounts of money to keep running 24/7. (For some reason while writing this it didn't occur to me you'd want the things actually living in a culture and incubator, which is cheap). There are DIY cell culture ingredients and most of the stuff is cheap. IIRC Fetal bovine serum is not and is kind of an awkward thing to purchase.

IIRC my interest in this was to get a skin cell and turn it into brain cells to put on a friggin robot with AI or something. I should stop acting like that's not still my interest because that's why I'm back here again.

My bio major friends have always said that a majority of their cultures just die for seemingly no reason, the things are hard to take care of and keep sterile. Divide the cultures up as much as possible.

I looked into making a DIY FACS (the fluorescent activated cell sorter) and that stuff is complicted. I forget what parts were too much for me. I never finished a project other than high voltage because I was doing so many projects simultaneously and reading mass amounts on them.

Never forget about SciHub




Oh.
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[*] posted on 30-8-2018 at 08:15


Quote: Originally posted by SupaVillain  

I also took a look at "apheresis" I believe it's called. This type of machine simply centrifuges the blood, and removes the stem cells, then puts the blood back into the patient. I think the problems with this were, some kind of apparatus for removing the stem cells being expensive, but I can't remember, they may just need to be siphoned out. Another problem MIGHT have been that these kinds of stem cells might not be able to turn into any cell other than various types of blood cells. It's been years since I've read into this stuff, sorry.


Yes, this is used to harvest hematopoietic stem cells for transplantation (they can be used to treat various blood cancers and blood disorders). This is called PBSC: peripheral blood stem cell collection. Ordinarily, there aren't all that many blood stem cells just floating about in your blood - they mostly occupy the bone marrow. However, treatment with filgrastim/recombinant G-CSF (granulocyte colony stimulating factor) causes increased production of blood stem cells - this is necessary before donating stem cells by PBSC.




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[*] posted on 30-8-2018 at 17:27


The following doesn't matter because hematopoietic stem cells harveted from peripheral blood only form blood cells.



Actually, when I looked one or two years ago, a FACS was incredibly hard to make and expensive. Now they've got cheaper 488nm laser pointers available on ebay and, after lookin at some laser hobbyist sites, and seeing the brand names they post, "sanwu" is another cheap option for around $100. Replacing the Argon Laser makes it much more possible.

Sanwu laser:
http://www.sky-lasers.com/sanwu-pocket-60mw-100mw-488nm-492n...

[Edited on 31-8-2018 by SupaVillain]




Oh.
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[*] posted on 31-8-2018 at 17:12


Culturing mammalian cells was considered the most challenging type of tissue culture. Then stem cells and iPSCs came along. You have set yourself quite the task. The equipment and reagents required are considerable.

The main pieces of infrastructure needed are:

Biological safety cabinet (sterile cabinet)
Incubator - temperature, humidity, CO2
Inverted microscope
Manual or automatic propipetter
Autoclave (if planning to reuse glassware rather than disposable plasticware)
Centrifuge
Liquid nitrogen storage for cell lines?

The main consumables are:
Plasticware (some combination of tissue culture treated flasks/plates, pipettes, bottles)
Media (including serum or prtein growth factors)
Carbon dioxide

I'm sure there will be some items I am missing. You may be able to find bargains second hand, diy some items or adapt protocols to use cheaper alternatives. For example, using 100% air and changing the buffer used rather than 5% CO2 and sodium bicarbonate buffer.

It is not only HEK293 cells that require liquid nitrogen for storage over the long term. All mammalian cells are stored this way. I only have experience at research universities but I have never encountered any limit on liquid nitrogen storage. There is a very definite economy of scale for liquid nitrogen storage though. Storage space is proportional to the dimensions cubed while the heat transfer and loss of liquid nitrogen is proportional to the square of the dimensions. Storing a few dozen vials in a small dewar would be very expensive and time consuming to maintain.

For context on the 488 nm laser for FACS comment, 488 nm is the excitation wavelength of some commonly used fluorophores - fluorescein, enhanced GFP, etc. You don't actually need any particular wavelength but you could save quite a bit of money on fluorophore labeled antibodies by paying extra for a common wavelength.

405 nm is another common wavelength used. This might be useful in an amateur context as it is the wavelength used by blu-ray and I think it will excite quantum dots. NurdRage has a video on making thse at https://www.youtube.com/watch?v=bNuoYm7Su4o
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