1. DIY Cell-based meat Roadmap & manual

2. Roadmap to DIY cell-based meat
DIY cell-
based meat
Edible cell scaffold
DIY cell culture
protocols
Consolidation of
“DIY cell culture”
Establishment of
communication
infrastructure &
communities
Control of differentiation
and tissue formation
Credits: @okgw_ )
Development of cell
culture hardwares
Cell culture device
with pumps
Implementation of
high-efficiency cell
culture hardware
Development of
tissue culture
hardware
Replace FBS
or egg yolk to
conditioned
medium
DIY serum
alternatives
Development of
automated cell
culture setups
Development of
growth factor
production protocols
based on coculture
Efficient cell culture protocols
Demonstration of
“DIY tissue engineering”

3. Overall steps of cell culture
Cell extraction P4
Cell centrifuge P5
Cell cyropreservation P13
Cell culture P10,11
Cell culture medium P6
Preparation of cell culture
environment P8
Cell passage P12
Observation of cells P9
Other cells and
prospects P14
Supplements preparation P7

4. Cell extraction
Incubate fertilized chicken egg for 8~12
days and extract cells from foetal tissues
Extraction from live tissues
Live cells are needed for cell culture
(the cells from meat and fish in supermarkets are most likely to be dead)
Cryopreserved cells Foetal cell extraction
Quickly defrost the cyrotube
containing the frozen cells using
40℃ water (cells die if done too slowly)
Purchased frozen cells or cells
prepared by methods on P13
Mince raw tissue, incubate at
37℃ for 40 minutes after adding
collagenase. Peel off cells from
the cellular tissues. (These are
the steps for cell dispersion.)
It is possible to use papain (which can
be purchased by commoners) instead,
but you may need to adjust incubation
time to 3-5 minutes as it is “strong”
https://drive.google.co
m/file/d/1dBDO9-4jvric
74R_3sPiiZyLpCbHZG
bh/view
http://zacmayu.hatenab
log.com/entry/2018/03/
04/235700
One of many
cell retailers

5. Cell centrifuge
Attach Falcon Tubes using duct tape.
If the tube weighs 30g, the centrifugal force is 5~6kg.
Fan = 800~1000rpm
20cm diameter: 143G
30cm diameter: 200G
Centrifuge of cell mass for about 1-3 minutes at 150-200G
The cells end
up here

6. The basal medium
https://www.slideshare.net/2co/diy-cult
ure-medium-manual-for-dmem-and-l15
“Sports drink culture medium” 「DIY-DMEM & L15 Leibovitz」
https://www.slideshare.net/2co/shojinmeat-proj
ect-open-source-cellular-agriculture-initiative
A brand of sports drink with pH
and osmotic pressures adjusted
could be mixed up to 60% with
DMEM without losing cell culture
efficiency.
Preparation of basal medium (DMEM and L15) solely
from accessible ingredients (food and food additives)
Mixing of sports drinks with DIY-DMEM and DIY-L15
media is a work in progress.
Mix 1X solution and 20x solution by 19:1, add glucose at 4.5g/L and bon apetit

7. Growth factors (growth hormones) are needed to signal cells to proliferate
Yeast extract + egg yolk 0.1%Serum, i.e. Foetal
Bovine Serum (FBS)
Co-culture / Conditioned media
Conventional very expensive
option used in university and
corporate labs - not available
in supermarkets, and has
safety concerns if for food.
One of the past experiments shows
yeast extract containing growth factors,
and missing ones can be sourced from
egg yolk for 5 days. The effectiveness
was comparable to FBS.
※More tests are needed to confirm
https://www.nicovideo.jp/w
atch/sm31104033
https://twitter.com/shoj
inmeat_jp/status/1104
214121375920129
Literature suggest mutual stimulation of cell
growth in the order of Placenta⇒Liver⇒
Muscle⇒Placenta
(Ohlsson C et al., 2009, Endocr Rev. Francis GL,
2010, Cytotechnology)
Fish cell growth can be induced by co-
culturing with kidney cells.
Medium supplements
GF’s

8. Setting up a cell culture environment
Hand-towel warmer hack DIY raspberry-pi
incubator
Reptile mat in a
styrofoam box
・Altering the AC in voltage from 100V to
30V reduces the operating temperature
from ~70℃ to ~38℃
・Portable drink warmer/cooler boxes
also do. If culturing fish cells, the room
temperature (controlled by an air
conditioner) is sufficient
(Credits; @okgw_ )(@earthlyworld)
・”Sotacubator”
・～$70
・On GitHub
・Open source
https://github.com/sotakan/RPi-Incubator
・$20～$40
・Beware of temperature
gradient and condensation
Drink boxes
also work

9. Atmospheric and pH control
CO2 forms by mixing
baking soda with citric acid.
at 3:1 molar ratio, 4:3
weight ratio. (25L CO2/mol)
DMEM mediums need ~5% CO2 to buffer the pH. L-15 Leibovitz medium does not
need it, but at a smaller buffer capability. This may be compensated by the quantity.
Sparkling water Baking soda + citric acid A lot of culture medium
By using a lot of medium, the pH
and concentration may perhaps
drift less with no atmospheric
control. ※Not confirmed yet

10. Contamination countermeasures in cell culture
Spraying 70% ethanol DIY Clean Bench Antifungal effect using egg white
Experiments often fail from mold and bacterial contamination.
Solutions: Don’t let mold pollens in, give the media “immunity”
Pharmacies are the most
likely place to find ethanol
in most countries
An air purifier combined with a
box or large plastic storage box
makes a DIY “clean bench”.
Egg white contain lysozyme, a
natural antimycotic. A mold-resistant
medium can be prepared by
mixing 5~10% of egg white.

11. Observation of Cells (microscopes)
Smartphone
Microscope
Standard school microscope DIY Inverted microscope
40X is enough to observe cells. An inverted microscope is ideal as it can see cells
stuck on the bottom of a cell culture petri dish.
From ~$10. May feel
difficulty focusing.
From ~$200. It is not an inverted
microscope - be careful not to let the
objective lens touch the medium
when focusing on the cultured cells.
Cells on the bottom
of the petri dish
A DIY inverted microscope using a junk
iPhone lens and a Raspberry Pi - may
have web connection to post on Twitter.
http://biohacker.
jp/c/BH222.html

12. Cell passaging
Detach cells from the
dish
Remove the medium and pour saline or
PBS. The cells are bound on the dish and
don’t easily get washed away.
Add 100μl of PBS or 1mL saline +
trypsin/EDTA solution and incubate for 1
minute at 37℃.
Wash cells
Pour out the red
culture medium and
wash the cells with
colorless PBS/saline
Passaging = Transferring of cells to a new dish and medium
If using scaffold
material...
If using cellular scaffold, cut the scaffold
into smaller pieces and place on new
scaffold. There is no need to remove cells
from the scaffold.
Dried konjac
scaffold
saline+trypsin
37℃ 1 min
cells come off by
tapping the dish
Transfer cells into
another vessel
Detach cells from dish by tapping the
bottom of the dish. Transfer into a test
tube, add 3ml medium and centrifuge.
move to a test
tube for
centrifuge

13. Cell Cryopreservation
Cool down to -70℃ using dry ice and ethanol or nail varnish (acetone).
Quickly freeze the cells (with cryopreservation agent) in the cyrotube.
After instant freezing, cell can be stored in a standard kitchen freezer.
- Freezing cells before formation of ice crystals, cell rupture and death.
Centrifuge cells
Add cryopreservation
agent
Freeze quickly
Centrifuge the mixture of trypsin, saline, cells - remove
the fluid component leaving the cells at the tip⇒P6
Add 1～2ml cryopreservation agent to the centrifuge tube.
Depending on the amount of cells obtained, cell mass can
be split to multiple tubes.
Cells pile up
at the tip upon
centrifuge
Cryotube
for cryo-
preservat
ion
The most important thing is to
protect cells from ice crystals
If slowly frozen, the sharp
ice crystals break the cell
membranes causing cell
death.

14. Varieties of cells & Future experiements
- Replace collagenase with papain
- Establish more stable DIY cell culture
protocols
- Mixing of sports drinks with DIY basal
media
- Further experiments with “0.05% egg
yolk as FBS replacement”
- Co-culture experiment to procure
growth factors
- Cell culture without 5% CO2 by using
large quantity of DIY DMEM,
Differences between species
Differences between cell types
The cell culture temperature may vary among species.
The efficiency may vary from “zero”, “not ideal” to
“sufficient” even if the temperature is not right.
Different cells need different growth factors. Many
types of cells need FBS, and liver cells also need
HGF to proliferate. Replacements and conditioned
medium (co-coculture) can be used to obtain growth
factors, even for brain nerve cells or iPS cell culture,
but this remains a future work.
birds &
mammals
DMEM
38℃
fish
DMEM
・L15
25℃
shellfish &
insects
L15
15~25℃
Future experiments

15. Participate in improving this manual!
https://docs.google.com/presentation/d/1cNedlmZcW7JOKB-gM
itL3aIix1YXd5gi0hhlYz1mVqQ/edit#slide=id.g7d2898c787_0_9
Source file openly
accessible and editable