Shojinmeat Project Clean Meat Initiative

Clean Meat Initiative

(Introduction)

What is cultured meat?

The technology(Questions session）

Social implications(Questions session)

“Cultured Meat” 2

“Cell culture school for kids” we organised

One of our student volunteers

“Shojinmeat Project” - Who we are 3

Collection of biohackers, science communicators, artists, student volunteers and so on, interested in cultured meat and cellular agriculture

Primary motivation in:Sustainability, culture and technology enthusiasm

Integriculture Inc. is the registered company to purchase reagents etc.

What/How we do 4

“Shojinmeat Project” develops cultured meat technology and engages in public communication.“Shojinmeat Project” is not affiliated with universities or corporations.

Low-cost culture media developed in private residence, materials from Rakuten/Amazon

Cell culture experiments in a secondhand incubator at Japanese “Indie Bio”, Leave-a-Nest Inc. lab

Self-funded, partly by SCIGRA, a science graphics & education service run by student volunteers.

Members participate in their respective expertise (experiments, gatherings, art projects etc.)

#Food Security #Food Miles

#Regulations

#Cooking

#Culture & thoughts#History#Food safety

#Life ethics#Animal welfare

#Regenerative medicine#Tissue engineering

#Bioreactor #Culture medium

#Business#LCA

#R&D

#Soc.&Econ.

Shojinmeat “Distributed Clusters” 5

#Global collab.

#Space

#Art


・Members tag themselves#R&D, #Cooking, #Ethical studies etc.

・One can even lead a cluster

・Clusters act independently, but occasionally share information

(Introduction)





8

“Meat”

From where?Meat is ~x40 more resource intensive

Lamb：~x50, Beef：~x40, Pork：~x20, Poultry：~x7

“Meat”←animals←feed, water, land

9

DeforestationFire-fallow cultivation Water shortage

“Meat”←animals←feed, water, land

10

Food as strategic resource and leverage

Food Security 11

“Insect Food” 12

MeatSoy etc. Dairy

MeatDairySoy etc. New alternative protein

Plants

Tofu

Algae Insects Biosynthetic Cultured

New protein source

“Meat & dairy produced in new ways”

Plant-based meat & dairy equivalent

Now

Future

“Alternative protein” and “meat alternative” 13

Tofu Plants

Algae

Insects

Biosynthetic

Quorn

Seitan

“Alternative protein” and “meat alternative” 14

Muscle cells Bioreactor

Culture medium

Processing

Cultured Meat 15

+Solution to 10000 year long ethical dilemma?

16Resources saved to 1/10~1/100

Research track record

1931　　　　1997　　　　2005　　　　2013　Goldfish culture@NASA

Netherlands funds research

Prof. Mark PostDemonstration

Concept known since 19c.

17

http://www.nicovideo.jp/watch/sm30099092

18Demonstration video on Niconico

Cellular AgricultureAgricultural products by cell culture

19Medical technology, Agricultural application

￥28,000,000Cultured burger, 200g

($260,000)

The Problem 20

Culture

Cook￥3000

($28)

2121Possible. Just expensive

Blame here

22Why so expensive

Cell culture is expensive

23Culturing of cells has beenoptimized for laboratory scale

Labs, Hospitals

Brewery, Petrochemical complex

Culturing of cells has been optimised for laboratory scale

Culturing of cells becomes industrial scale

24Conditions and purposes change→Entire process needs a re-design

No 100mm dish but use 25㎥ tankNo gloves but do tank sterilisationNo pipettes but use pipelines If tanks are sterilised, no antibiotics needed

Conditions and purposes change→Entire process needs a re-design

25

・Meat production is very resource intensive・Lack of resources leads to resource war・Cultured meat is a potential solution・Possible to make, but expensive・Cell culture methodology needs redesign

What is cultured meat? (summary)26

(Introduction)





2.System

1.Media

3.OperationWhat needs redesign? 28

$15 $450 $410

Medium + Foetal bovine serum(FBS) + Growth factoramino acid, minerals,sugar, vitamines

Albumin, insuline, transferin

cell growth factor

Culture medium 29

= Cow blood

Standard DMEM(FBS10%) 500ml

$30,000 for 100g 500ml of culture medium produces 200mg of cells

Cost of culture media

(yen/JPY)

30

Block et al., 1996

DMEM　 450ml　￥1125FBS 50ml ￥4900Non essential amino acid　￥140

HGF 40ng/ml ￥78000 (20µg)　EGF 20ng/ml ￥700 (10µg)

￥84865($8000)

Culturing 100g of liver cells cost $400,000

Liver cell culture for therapy 31

SugarAmino acidsVitaminsMinerals

AlbuminBufferInsulinTransferin

Growth factorsSurvival factors

Essential medium

Foetal bovine serum

Signal compounds

Expensive… :-(“Mad cow”?Viruses?Expensive and unstable supply

Gospodarowicz D and Moran JS, 1976, Annu Rev Biochem Eagle H, 1959, Science

Expensive for what’s actually in

＝

Bottlenecks in culture media 32

Our research direction 33

・Biohack.Do things do-able without privilege

・Start with something radically low-cost Such materials become animal-free by economics

・No jumping to making meatFind a cheap way to do it first.

Yeast extract as FBS alternative? 34

Commercially available:“Yeast extract for animal cell culture”

(Sheffield Bioscience Ltd.)

Dried yeast (dog food)

Phosphate buffer & saltPapain, 65℃, stir, ~24hto “digest” yeast

Filtered before use in cell culture

pH7.4No bacterial contaminationPreparation cost: 10¢/L

Myoblast cell response 35

The trial solution showed signs of L6 growth and multiplication. ⇒”Yeast extract (dog food) is a potential FBS alternative.”

NO

FB

SC

ontr

ol

(with

FB

S)Mouse L6 Myoblast cell density (qualitative)Red ~ Yellow ~ Blue(zero)

No DMEM

w/ DMEM

No DMEM

w/ DMEM

Conc. yeast extract

Dil. yeast extract

HeLa cells in yeast extract medium 36

HeLa cells in sYE (yeast extract) multiplied at 80% of that in FBSHeLa cells in sYE medium grew in diameter more than in FBS

Cel

l cou

nt (x

10e5

)(HeLa = Human cervical cancer cell line)

Day0 Day7

FBS 10%

sYE 10%

sYE 10% +FBS 10%

293T cells in yeast extract medium 37

sYE is capable of culturing 293T cells, which have relatively strict requirement for FBS to thrive

Cel

l cou

nt (x

10e5

)(293T = Human Embryonic Kidney cells)

Day0 Day7

FBS 10%

sYE 10%

sYE 10%+FBS 10%

10％ FBS

10％ sYE

10% FBS10% sYE

Cellular self-organisation in sYE 38

96-well suspension culture plate

Mouse myoblasts were multiplied to 1.0E5 in standard FBS medium Cells were placed under suspension culture condition in sYE medium for 4 days⇒Self-organised into a 3-dimensional aggregate of 0.3~0.5mm diameter

The limits 39

Species Cell identity sYE conc. Result

Human HeLa Human cervical cancer 10％ Successful (but for how long?)

Human Human Embryonic Kidney 293T 10％ Successful (same as above)

Mouse Germ cell 10％ Unsuccessful

Mouse Ovarian somatic cell, primary 10％ Successful (but for how long?)

Mouse Hepatocyte, primary 10％ Successful (same as above)

Mouse Fetal cell, primary 10％ Successful (same as above)

Mouse Myoblast, primary 10％ Initially successful, but cell division halts after ~3gen.

Mouse Embryonic stem cells 10％ Unsuccessful

Yeast extract do have limits.Unsuitable for undifferentiated cells.

2.System

1.Media

3.OperationWhat needs redesign?

Breaking the limits 41

How do body fluids acquire albumin, growth factors etc.?⇒Intercellular interactions

Ohlsson C et al., 2009, Endocr RevFrancis GL, 2010, Cytotechnology

HGFEGF

TGFβAlbumin

Adrenalin

Coculture system (proto-prototype)

PCT/JP2016/067599Japan pat. 2016-568716

Conditioning of culture media by coculture 42

Control (0%) 10% conditioned medium

25% conditioned medium

50% conditioned medium

Cou

nt o

f cel

ls o

f all

size

s,

rela

tive

to th

e co

ntro

l gr

oup

Secondhand medium is effective!

mouse placental cells

dishes with Day-12 foetal liver cells in FBS 10% medium

7 Days

Transfer culture medium

Low-cost liver cell culture 43

DMEM　 450ml　￥1125Non essential amino acid　￥140FBS 50ml ￥4900HGF 40ng/ml ￥78000 (20µg)EGF 20ng/ml ￥700 (10µg)

￥84865(€750~)

Liver cell aggregate on collagen scaffoldCultured by coculture in sYE medium

DMEM　 450ml　￥1125sYE 50ml ￥182

￥1307 (€12)

The full potential of “sYE in coculture” 44

DMEM　 450ml　￥1125sYE 50ml ￥182

￥1307 (€12)

”DMEM” 450ml　￥10sYE 50ml ￥1

￥11 (€0.10)

SugarAmino acidsVitaminsMinerals

Essential medium =

from algae?

MediumGO 45

Huge variety on streets to choose from!

The cost of media for 100g of cells 46

Current method cultures only 1 layer of cells on the bottom.

Improvements in culture efficiency 47

Scaling as it is... 48

How cultured burger was made in 2013⇒$260,000

Suspension culture Cellular scaffolds

More efficient methods 49

Either way is called “3D culture”, not bound on 2D dish

FIRST Project by Prof. OkanoSystem integration for commerci-alisation of regenerative medicinehttps://www.youtube.com/watch?v=g7lyTsxbt2U

Suspension (3D) culture uses less medium

Culture media

Culture media

27 Litres

9 Litres

What it takes to grow 1B iPS cells

Efficiency by suspension culture 50

Cellular scaffold also achieves the advantage of 3D culture

Sponge collagen scaffolds Liver cells on scaffolds

3D culture by cellular scaffolds 51

Edible scaffolds i.e. collagen, chitosan, chitin, arginate, cellu- lose, polysaccharides

Simulate fibre and meat texture

Moulds shape in mm or bigger scales

Other benefits of scaffolds 52

Integration of all elements into one scalable system

~0.1g scale ~10g scale ~100kg scale future pilot plant

All in one system: sYE, scaffold, suspension 53

PCT/JP2016/067599　jp-pat file# 2016-568716

All in one system: sYE, scaffold, suspension

technologically demanding“Is it tasty?” 55

“Ground meat” “Meat sheets” “Steak”At least it’s meat Juicy tastes Meat textureMade by muscle +Fat cells +Blood vessels,+fat cells Regen. MedicineProven ※though expensive Scaffolds

2.System

1.Media

3.OperationWhat needs redesign? 56

Not at all big enough for food

57Current biggest cell culture

Plant engineering 58

How is temperature controlled?

Mixing method?

Pipeline diameter & flow rate?

Sterilization method & frequency?

How are filters cleaned?

Plant engineering - what exactly? Speculative fish meat culture plant

・Resource requirement from ‘cradle to the grave’

・LCA is necessary for plant engineering

Life cycle assessment (LCA)

“Meat brewery” 61

Farm high-rise 62

Mars Colony 63

Orbital Zero-G Farm 64

(Introduction)





Would vegetarians eat?Is it Halal?Goes with Buddhist “nonviolence”?Animal welfare?Consumer acceptance?At the end, is it tasty?

Social and cultural implications 66

A. Animals, religion B. Food security C. Food safety

D. Env. footprint

EU/US：　A~D > C > BE.Asia：　B~C > A > D

Regional differences in agenda 67

MENA & India: i.e. Halal/Kosher 68

・Yehuda Shurpin, a Jewish cleric: “If the original cell is “Kosher”, so would be the cultured meat, but let’s wait until technologies mature.”・Abdul Qahir Qamar of Jeddha Islamic Academy: “food from animal secretes - isn’t it just yoghurt?”・Holy Cow for for-profit operations? NO, killing or welfare isn’t the issue・Pork is “unclean” because of its lifestyle and habitat? ….or the DNA?

Utilitarian & Animal welfareBuddhist “nonviolence” commandmentGeneral consumer acceptance?“New Ethics” controversy in 2040

Selected topics 69

Personal beliefs than religions

・Vegetarians・Pescetarians・Vegans are the most common

Western vegetarianism 70

“Max. happiness for the max. number of sentient beings”※Not “ends justify means”, but an guide to arguments over what’s ethical

“Only 1 death is better than 5”...?May be so in very short term, but...

What if the person is your brother? A society where abandoning of family is justified, would it be a good one?

or in another example,If “killing for public good” becomes the norm, no one is there to stop dictators - would it really do “max. happiness for max. number of people?　”Long-term total happiness must be discussed”

Classic “Runaway trolley problem”As it is, 5 will die, but if switch, 1 dies. Would you switch?

Practical ethics: “Utilitarianism” 71

“Animal welfare” deduced from utilitarianism 72

All interests are equal: “1 person, 1 vote”No being’s happiness is more important than another

◆Are animals capable of experiencing happiness?◆Should an animal count as one sentient being?

In “animal welfare”, speciesism is dismissed & animals count as beings that can experience happiness

⇒From a utilitarian point-of-view, “making a sentient being suffer is unethical”

Acknowledges health and environment, but mainly animal welfare and ethical

“Animal welfare” based on utilitarianism

Reasons for vegetarianism 73

Announced in 2008 ⇒ taken down on 2014: technology wasn’t ready

$1M Award by PETA 74

Interests inAnimals⇒↓Veggie

YES NO

YES Vegans, mainstream Western vegetarians

Opinions divided

For-health veggies, Pescetarians

“Not for me”...?But beef meat fish fat “healthy meat” maybe?

NO Animal welfare organisationsPet owners, animal lovers

All-out YES

General consumers

What is it? GMO? Zombie meat?

Opinions and positions 75

”Religion”←Not utilitarianism or other ethics

“Ethical” aspects of cultured meat don’t necessary translate into East Asian religious (i.e. Buddhist) importance.

East Asian vegetarianism 76

Cuisine for for zen practitioners. Its preparation is also part of zen practice.

・Common name for “Buddhist cuisine”・Uses only local produces・Donated food - use without waste・No use of meat and several specific vegetables.※If meat is donated, it is cooked and consumed.

Cultured meat doesn’t equal “shojin ryori” solely on the grounds of “because it hasn’t killed” - it must carry more religious meanings.

“Shojin ryori” Buddhist cuisine

Holy text↓

Rules↓

Daily life

Religious rules in Abrahamic religions

RulesMiddle way

Daily life

Holy texts

・Middle way > Rules・Making religious rules as something “absolute” goes against the middle way.

・Nonviolence is one of the rules

・Monks eat meat, if donated.

・Cultured meat would be seen as a kind of “tofu (fake meat)” Use it as a reference

in making decisions

Mentions

Religious rules in Eastern religions

“The Middle Way”

Before 7th century：Eating meat was common. People just had to eat whatever was in hand.

675c. Imperial decree of “No Killing (of animals)”To direct labour force to rice production and put a stop to local animal-sacrifice rituals & reinforce imperial authority※Newly arrived Buddhism was used as justification

Meat avoidance continues till 19th c. and commoners only started eating meat around 1900 c.

Meat in historical Japan

Totals half billion? Region-specific

Found more in upper caste, which even funds cultured meat research

Hinduism doesn’t explicitly forbid, but adherents choose to avoid meat.

Current Indian PM is one.

Vegetarianism in India 80

Guardian, 2014/09/11

(2014/08/05)

Public opinion is fluid due to lack of information on the subject.

Very fluid

General consumer acceptance

NO YES

(Transient) issues upon R&D：・FBS production is not “cruelty-free”

“Unavoidable” issue：・Extraction of cells

Likely to be solved in the futureMay pose an issue during R&DCan bovine foetus feel pain?

It may still inflict some pain.Will animals still be chained?Genetic selection of animals for the sake of “tasty” - is it eugenics?

Ethical issues due to technological immaturity

Then what if on 2040, cultured meat takes up 30% market share and more people stop consuming live animals for meat?・Uncontrollable ”hate campaign” against traditional farmers?・Trade ban of “real” meat by WTO on animal rights grounds?

Why are animal experimentation, Japanese whaling and Chinese cat/ dog consumption is problematised far more than factory farming? ⇒Because they are “remote things” for the protesters.

Possibility of “New Ethics”

・Can countries with meat culture technology blame others for animal abuse?

・”Patent infringement to save animals in countries that don’t have the technology”

Case study: Generic HIV drug lawsuit:

An Indian pharmaceutical company allegedly infringed retroviral drug patent to manufacture generic HIV drugs, because the original drugs by Western pharmaceutical companies were too expensive for people in poor African countries.

After high-profile court-martials, the Indian company won the case on humanitarian basis.

Patent war under “New Ethics”

Global meat$1.5T

Global beef$0.7T

Japan$50B

Toward “price parity”

Startups

Funds

NPOs

Key start-ups and NPO’s

Members participate in their respective expertise (experiments, gatherings, art projects etc.)

#Food Security #Food Miles

#Regulations #Cooking

#Culinary culture#History#Food safety

#Life ethics

#Animal welfare#Regenerative medicine#Tissue engineering

#Bioreactor #Culture medium

#Business

#LCA

#R&D#meat culinary culture#Soc.&Econ.


Acknowledgements 87

Lab space, various technical & management advices

(Ms. Murata’s private residence)

Members & Volunteers

Dr.Komatsu Research Office

“Science Agora” event organization

“Homebase”meeting & event space

Support for our “Cultured meat fanzine” distribution

Niconico science community

Miyo-sanFrom ”Myosin”

age.20・164cm

Chemical engineering student intern at Mars Huygens Crater cellular agriculture plant

Aco-chanFrom ”Actin”

age.13・149cm

Helps elder sister Miyo as part of extracurricular activity offered by Mars Colonists Middle School.

Miyo & Aco

Miyo-san (Chibi)

Aco-chan (Chibi)

Contacts 93

http://[email protected]



Scientific VR, 3D printing and rendering service -funds Shojinmeat Project

http://lne.stSeed accelerator

http://shojinmeat.com


mailto:[email protected]


http://integriculture.co

http://integriculture.co





