Plant Protoplast Microfluidics

[Sebastian Cocioba]

Hi Everyone!

I've been toying with some designs for a protoplast culture and PEG transformation chip and had a few questions:

1. Some of the only papers citing tobacco protoplast cultures and microfluidics have a design that consists of a wide channel which at one end have a sieve of posts a few microns in diameter and a few micros apart. This collects the cells, which range from 40-70um while replacing the media. This would cause an aggregate as the sieve fills and would damage cells...i think.  Do you think an laser printer set to 1200 DPI can print a grid 20-30 microns apart? The post size won't matter as long as the sieving space is tight.

2. How is oxygenation and media recirculation done in bacterial culture chips? Do they just circulate the media without losing cells somehow?

3. Lets say I get protoplasts to make microcolonies, how (aside from cutting open the PDMS culture chamber) would I get them out to replate?

The whole idea of this chip is to repeatedly perform PEG fusions and naked plasmid uptake experiments on protoplasts and regenerate them. There is a lot of manual skill and tricks involved in PEG transformation so I want to take luck and dexterity out of the equation. Any advice would be fantastic.

4. Know any literature on standardized or good design practices for general microfluidics fabrication? I'm rockin PDMS-glass chips made using shrinky dinks and if it works Polyolefin shrink wrap chips too. I found a book that covers all the theory and fluid dynamics behind the curtain which is great but little on channel layout theory/advice/style.

5. Has anyone made valves using the shrinky dink method? The channels end up rounded which isn't the ideal geometry for the control layer.


Thanks in advance!

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D

[Nathan McCorkle]

On Thu, Dec 5, 2013 at 11:13 AM, Sebastian Cocioba <scoc...@gmail.com> wrote:
> Hi Everyone!
>
> I've been toying with some designs for a protoplast culture and PEG
> transformation chip and had a few questions:
>
> 1. Some of the only papers citing tobacco protoplast cultures and
> microfluidics have a design that consists of a wide channel which at one end
> have a sieve of posts a few microns in diameter and a few micros apart. This
> collects the cells, which range from 40-70um while replacing the media. This
> would cause an aggregate as the sieve fills and would damage cells...i
> think.

Maybe, how did the paper(s) get around aggregation and/or damage?
Simply not loading so many cells so an aggregate won't be possible?

> Do you think an laser printer set to 1200 DPI can print a grid 20-30
> microns apart? The post size won't matter as long as the sieving space is
> tight.

First some basic arithmetic, let's say 1 inch is 25400 microns, then
25400/1200 = 21.16666

So that's 1 dot per 21.66 microns.

Then you have to figure out your minimum and maximum dot sizes. For a
laser printer this is the toner particle size, from wiki:
"Originally, the particle size of toner averaged 14–16 micrometres[1]
or greater. To improve image resolution, particle size was reduced,
eventually reaching about 8–10 micrometers for 600 dots per inch
resolution."
and
"Toner has traditionally been made by compounding the ingredients and
creating a slab which was broken or pelletized, then turned into a
fine powder with a controlled particle size range by air jet milling.
This process results in toner granules with varying sizes and
aspherical shapes. To get a finer print, some companies are using a
chemical process to grow toner particles from molecular reagents. This
results in more uniform size and shapes of toner particles. The
smaller, uniform shapes permit more accurate colour reproduction and
more efficient toner use."

But there are other factors that limit the resolution: how many
particles the printer calls 'light' or 'dark' in its image settings;
how light or dark the pixel is in the data being sent to the printer;
the odds that you can actually place (1) 5 micron particle in the 21.6
micron pixel; are the particles round, oblong, random spiky looking
crystals?

See one method here:
Ohttp://diyhpl.us/~nmz787/pdf/Rapid_and_inexpensive_fabrication_of_polymeric_microfluidic_devices_via_toner_transfer_masking.pdf
http://diyhpl.us/~nmz787/pdf/Rapid_and_inexpensive_fabrication_of_polymeric_microfluidic_devices_via_toner_transfer_masking__Supplement.pdf

>
> 2. How is oxygenation and media recirculation done in bacterial culture
> chips? Do they just circulate the media without losing cells somehow?

You could do that, with say a nanoporous membrane:
http://www.simpore.com/products.html

or maybe a decent field of posts to block cells if they're big enough,
otherwise I've seen the gas permeability of PDMS used, so it would be
a parallel-plate membrane setup, cross-flow counter-flow or cocurrent
flow:
https://www.housing.ou.edu/content/dam/CoE/CBME/Undergraduate_Lab/CHE3432/PDF_files/Chapter13MembraneSeparation.pdf

A practical guide to microfluidic perfusion culture of adherent mammalian cells
http://www.rle.mit.edu/biomicro/documents/lykim_LOC2007.pdf

> 3. Lets say I get protoplasts to make microcolonies, how (aside from cutting
> open the PDMS culture chamber) would I get them out to replate?

Open a big valve that was previously shut? Backflush?

> The whole idea of this chip is to repeatedly perform PEG fusions and naked
> plasmid uptake experiments on protoplasts and regenerate them. There is a
> lot of manual skill and tricks involved in PEG transformation so I want to
> take luck and dexterity out of the equation. Any advice would be fantastic.

Get a 1080p webcam fitted to your microscope. Logitech makes the C615
which is ~$50, any dSLR that feature electronic shutters (aka 'full HD
video', where the physical shutter doesn't actuate) would be even
better. Connect them to a laptop or raspberryPi (since you need some
GPIO anyway for controlling valves and pumps and lights/filterwheels,
etc), then you can at least monitor your cells easily and record
images/videos, and later maybe start to automate with machine vision
(i.e. rinse with PEG until cells reach detected diameter).

> 4. Know any literature on standardized or good design practices for general
> microfluidics fabrication? I'm rockin PDMS-glass chips made using shrinky
> dinks and if it works Polyolefin shrink wrap chips too. I found a book that
> covers all the theory and fluid dynamics behind the curtain which is great
> but little on channel layout theory/advice/style.

These are pretty good:
www.kni.caltech.edu/foundry/basic_rules.html

> 5. Has anyone made valves using the shrinky dink method? The channels end up
> rounded which isn't the ideal geometry for the control layer.

The toner-transfer paper I linked earlier says that the horizontal is
traded off for vertical, so channel volume remains constant.

[Nathan McCorkle]

Oh, I found this too, which shows printing toner on transparency
plastic then embossing with PDMS can achieve 6-12 microns (STL, single
tone layer or DTL, double tone layer)

Toner and paper-based fabrication techniques for microfluidic
applications (Review)
http://diyhpl.us/~nmz787/pdf/Toner_and_paper-based_fabrication_techniques_for_microfluidic_applications.pdf

I guess the link (in my previous email) was on toner-transfer with
etching, so there
was also diffusion going on in that to reduce resolution.

There's this too (which is ref 17 of the last link)
http://diyhpl.us/~bryan/papers2/microfluidics/Rapid%20fabrication%20of%20microfluidic%20devices%20in%20poly(dimethylsiloxane)%20by%20photocopying.pdf

and ref 18 (which is where it get's the STL and DTL data)
http://diyhpl.us/~bryan/papers2/microfluidics/A%20Dry%20Process%20for%20Production%20of%20Microfluidic%20Devices%20Based%20on%20the%20Lamination%20of%20Laser-Printed%20Polyester%20Films.pdf

So seems like your idea is feasible and is worth finding someone with
a laser printer and laminator!

--
-Nathan