DIY Cell-based meat Roadmap & manual
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”
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
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”
One of many cell retailers
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
The basal medium
“Sports drink culture medium”
「DIY-DMEM & L15 Leibovitz」
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
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
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
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)
・$20～$40
・Beware of temperature gradient and condensation
Drink boxes also work
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
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.
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.
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
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.
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- preservation
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.
Varieties of cells & Future experiements
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
Varieties of cells & Future experiements
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
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Source file openly accessible and editable